NDLI: Expression, purification and immobilization of the intracellular invertase INVA, from Zymomonas mobilis on crystalline cellulose and Nylon-6

Content Provider	Springer Nature Link
Author	Ángeles Calixto Romo, María Santiago Hernández, José Alejandro Vallejo Becerra, Vanessa Amaya Delgado, Lorena Carmen Montes Horcasitas, María Hidalgo Lara, María Eugenia
Copyright Year	2008
Abstract	This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K m and V max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V max increased up to 5.7-fold, following immobilization, whereas K m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.
Starting Page	1455
Ending Page	1463
Page Count	9
File Format	PDF
ISSN	13675435
Journal	Journal of Industrial Microbiology and Biotechnology
Volume Number	35
Issue Number	11
e-ISSN	14765535
Language	English
Publisher	Springer-Verlag
Publisher Date	2008-08-20
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Enzyme immobilization Invertase Nylon-6 Sucrose Zymomonas mobilis Microbiology Bioinformatics Biotechnology Genetic Engineering Inorganic Chemistry Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Bioengineering Applied Microbiology and Biotechnology Biotechnology

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